Induction of immune tolerance

ABSTRACT

Methods of inducing immune tolerance by administering an immunosuppressive agent and a compound represented by Formula (I) are disclosed:  
                 
 
     Additionally methods of suppressing an immune response by administering an immunosuppressive agent and a compound represented by Formula (I) are disclosed. Further disclosed are methods for treating autoimmune diseases by administering an immunosuppressive agent and a compound represented by Formula (I). The variables of Formula (I) are described herein.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/428,328, filed Nov. 21, 2002, the entire teachings ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Autoimmune disease results when a subject's immune system attacksits own tissues often resulting in severe morbidity or death. Examplesof such diseases include type 1 insulin dependent diabetes, multiplesclerosis, rheumatoid arthritis and inflammatory bowel disease.

[0003] The immune system also can cause illness as a consequence of itsreaction to a transplanted organ, tissue or cell, where the organ,tissue or cell is recognized as foreign. Typically the transplantedorgan or tissue is recognized as foreign by the host and infiltrated bythe host immune cells, which then carry out their effector function anddestroy the transplant. Typical treatments to help suppress the immuneresponse that is elicited by foreign transplant include immunosupressivedrugs such as corticosteroids, cyclosporin A, rapamycin and FK506 orantibody therapies such as anti T-cell antibodies. These therapies arenot specific and typically must be administered for the life of thepatient. Additionally, these therapies have undesirable side effectssuch as high blood pressure, renal failure, lowered resistance toinfection and fever. The costs of those treatments over a lifetime isalso quite expensive.

[0004] In response to the aforementioned problems associated withimmunosupression therapies, there exists a clear need for moreefficacious and less problematic therapies to treat autoimmune diseasesand organ or tissue rejection.

SUMMARY OF THE INVENTION

[0005] It has now been found that compounds of Formula (I), which aredescribed below in detail, and certain immunosupressive agents (e.g.,rapamycin or anti CD40L monoclonal antibody) act synergistically toinhibit transplant rejection and can even induce specific immunetolerance towards transplanted tissue. For example, administration of asub-therapeutic dose of rapamycin together with Compound 1 to mice withheart transplants suppressed transplant rejection for at least 100 days(see Example 1). Rejection did not occur, even after treatment withthese drugs was terminated. Furthermore, a second heart transplant fromthe same donor strain of mice was accepted, while a second hearttransplant from an unrelated donor was rejected (Example 3). Theseresults indicate that this combination therapy induced specific immunetolerance towards the transplanted organ without causing non-specificimmune ablation. In addition, anti-CD40L monoclonal antibody andCompound 1 were also found to act synergistically to inhibit hearttransplant rejection in mice. Mice treated with this combination showedno sign of rejection after 100 days (see Example 2). Based on theseresults methods of inducing immune tolerance in a subject in which thereis an undesired immune response, methods of preventing, inhibiting orsuppressing rejection of transplanted organs and methods of treatingautoimmune diseases are disclosed herein.

[0006] In one embodiment, the present invention provides a method ofinducing immune tolerance in a mammal in need of such treatment. Themethod comprises the step of administering to the mammal an effectiveamount of an immunosuppresive agent and an effective amount of acompound of Formula (I):

[0007] R₁ is a substituted or unsubstituted aryl group or a substitutedor unsubstituted alkyl group.

[0008] R₂ is an optionally substituted aralkyl group or an alkyl groupsubstituted with —NR₅R₆.

[0009] R₃ is a substituted or unsubstituted alkyl group or a substitutedor unsubstituted aryl group.

[0010] R₄ a substituted or unsubstituted alkyl group or a substituted orunsubstituted aryl group.

[0011] R₅, and R₆ are independently selected from a substituted orunsubstituted alkyl group or a substituted or unsubstituted aryl groupor R₅ and R₆ taken together with the nitrogen to which they are attachedare a non-aromatic heterocyclic group.

[0012] In yet another embodiment the present invention provides a methodof inhibiting rejection of a transplanted organ, tissue or cell in amammal. The method comprises the step of administering to the mammal aneffective amount of an immunosuppressive agent and an effective amountof a compound of Formula (I).

[0013] In yet another embodiment the present invention provides a methodfor suppressing (or inhibiting) an immune response in a mammal byadministering an effective amount of an immunosuppresive agent and aneffective amount of a compound of Formula (I).

[0014] In another embodiment the present invention is a compositioncomprising an immunosuppressive agent and a compound of Formula (I). Thecomposition can be used in therapy, for example, to induce specificimmune tolerance in a subject in need thereof, to inhibit organ, tissueor cell transplant rejection or to treat autoimmune disease. Typically,the composition comprises an effective amount of both agents. Thecombination can also be used for the manufacture of a medicament forinducing immune tolerance, treating organ, tissue or cell transplantrejection rejection and/or autoimmune disease.

[0015] The immunosupressive drugs used in the combination therapydescribed herein synergize and therefore are highly effective insuppressing organ transplant rejection. In many instances, thecombination induces specific immune tolerance towards the transplantedtissue. As a consequence, treatment can be terminated once tolerance hasbeen induced, thereby eliminating the need to remain on a lifelongregimen of immunosuppressive drugs. In addition, cessation of theimmunosuppression allows the immune system to react with full strengthagainst encounters with new antigens. Because the component processes oftransplant rejection are shared with a number of autoimmune diseases(e.g., multiple sclerosis, juvenile onset diabetes, inflammatory boweldisease, lupus and rheumatoid arthritis), the disclosed combinationtherapy is expected to be useful in treating autoimmune disorders aswell. Moreover, the disclosed methods can be used to induce tolerance toother types of antigens, including therapeutic agents that wouldotherwise be recognized as foreign by a subject's immune system, e.g.,proteins, protein fragments, viral vectors used for gene therapy and thelike.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 is a graph showing the survival rate of mice over time (indays) after receiving heart transplant. The mice were divided into fourtransplanted groups: 1) mice without treatment; 2) mice treatedsubcutaneously with 0.3 mg/kg/day of rapamycin for three days followingthe transplant; 3) mice treated intraperitoneally with 75 mg/kg/day ofCompound 1 for two weeks following the transplant; and 4) mice treatedsubcutaneously with 0.3 mg/kg/day of rapamycin for three days andintraperitoneally with 75 mg/kg/day of the compound of Compound 1 fortwo weeks following the transplant. FIG. 1 shows that the Compound 1synergizes with rapamycin such that cardiac allograft rejection wasprevented for all recipients for at least 100 days in this mousetransplant model.

[0017]FIG. 2 is a graph showing the survival rate of mice over time (indays) after receiving heart transplant. The mice were divided into fourtransplanted groups: 1) mice without treatment; 2) mice treated with asingle dose of anti-CD 154 monoclonal antibody (250 μg/mouse)immediately following the transplant; 3) mice treated with 75 mg/kg/dayof Compound 1 for two weeks following the transplant; and 4) micetreated with a single dose of 250 μg of anti-CD154 monoclonal antibodyimmediately following transplant and 75 mg/kg/day of Compound 1 for twoweeks following the transplant. FIG. 2 shows that Compound 1 synergizeswith anti-CD154 monoclonal antibody such that cardiac allograftrejection was prevented in all recipients for at least 100 days in thismouse transplant model.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The disclosed invention is a combination therapy of animmunosuppressive drug and the compounds of Formula I.

[0019] The combination therapy has been found to synergisticallysuppress tissue transplant rejection and typically to induce immunetolerance to the transplanted tissue. Tolerance towards other types ofantigens, e.g., therapeutic proteins and viral vectors used in genetherapy, can also be induced. Because the effector mechanisms used bythe immune system during tissue transplant rejection are broadly sharedwith and central to the pathology occurring in a number of autoimmunediseases (e.g., multiple sclerosis, juvenile onset diabetes,inflammatory bowel disease, lupus and rheumatoid arthritis), thecombination therapy is also therapeutically useful for treating manyimmune and/or inflammation based diseases.

[0020] The term “immunosuppressive agent” refers to a drug which is ableto suppress an immune response and is preferably a drug which suppressesa T cell mediated immune response. Immunosuppressive agents have beencommonly used following tissue transplants to suppress acute rejectionof the transplanted tissue. Examples of immunosuppressive agentsinclude, but are not limited to, anti-lymphocyte antibodies (e.g.,anti-lymphocyte serum, anti-CD3 monoclonal monoclonal antibody andanti-CD4 monoclonal antibody), rapamycin, FK506, cyclosporin A,corticosteriods, azaihioprine, mycophenolic acid, cytokines,lymphotoxins, inhibitors of costimulatory molecules (e.g., anti CD40Lmonoclonal antibodies and anti co-stimulatory T-lymphocyte antigen 4immunoglobulin (CTLA4 Ig)). Rapamycin and inhibitors of co-stimulatorymolecules such as anti-CD40L monoclonal antibody are most commonly usedin the disclosed combination therapy.

[0021] The term “inducing immune tolerance” means rendering the immunesystem (preferably the T cells of the immune system) unresponsive orless responsive to a particular antigen without inducing a prolongedgeneralized immune deficiency. The term “antigen” means a substance thatis capable of eliciting an immune response. For autoimmune disease,“inducing immune tolerance” means rendering the immune systemunresponsive or less responsive towards autoantigen or other antigenthat the host recognizes as foreign and against which the host isgenerating an autoimmune response. Induction of this type of tolerancecan be used to treat autoimmune disease. For transplantation, “inducingimmune tolerance” means rendering the immune system unresponsive or lessresponsive towards the antigens on the transplant, the transplantedorgan, tissue or cells.

[0022] The disclosed methods can be also used to induce immune tolerancetowards other types of antigens which evoke an undesired immuneresponse. For example, the safety and efficacy of a wide variety ofpotential therapeutic agents may be compromised by undesired immuneresponses to the agent being administered. Inducing tolerance to theseagents by the disclosed methods will prevent clearance and destructionof these agents by the immune system and enable them to carry out theirtherapeutic function. Examples of such agents include therapeuticproteins (e.g., antibodies, enzymes and the like), protein fragments andserums that are obtained from a species different than the host or whichotherwise comprise antigen that are recognized as foreign by the host'simmune system. Other examples include certain agents used in genetherapy, e.g., viral vectors. “Inducing immune tolerance” to theseantigens means rendering the immune system unresponsive or lessresponsive to the antigen without inducing a prolonged general immunedeficiency.

[0023] Other types of antigens which evoke undesired immune responsesinclude certain allergens or venoms, particularly when the immuneresponse evoked by these antigens is mediated by T cells. The disclosedmethods can be advantageously used to induce tolerance towards antigensof these types. “Inducing immune tolerance” to venoms and allergensmeans rendering the immune system unresponsive or less responsive to theantigen without inducing a prolonged general immune deficiency.

[0024] As described above, the disclosed methods can be advantageouslyused to treat a mammal with an organ, tissue or cell transplant. Themost common type of transplant is an allograft, which is a graft betweenmembers of the same species. Cells, tissues or organs (or parts thereof)which are typically transplanted between members of the same speciesinclude, but are not limited, to heart, lung, kidney, liver, pancreas,pancreatic islets, brain tissue, cornea, stomach, bone, bone marrow,muscle, intestine, bladder, skin and stem cells. Optionally, thetransplanted tissue or organ is bio-engineered, e.g., when thetransplanted tissue or organ is grown from a stem cell or other type ofprecursor cell(s). Bio-engineered tissue or organ can be grown outsideof the body and transplanted directly into the host. Alternatively, theprecursor cells or immature organ or tissue is transplanted into thehost to grow and mature.

[0025] The tranplanted organ, tissue or cell(s) can also be a xenograft,i.e., the donor is a member of a species different than the recipient.Xenografts are advantageously used with a bio-engineered tissue ororgan, which, instead of being transplanted directly into the recipientin need of the tissue or organ, can be transplanted into a surrogatehost such as non-human mammal until a suitable human recipient in needof the bio-engineered tissue or organ is identified. Alternatively, thetissue or organ can be transplanted into the surrogate to allow thebio-engineered tissue or organ to mature. Use of surrogate hosts may bepreferred in instances where further development of the tissue or organis required before transplantation into a human recipient. In anotheralternative, a xenograft is used when a suitable allograft donor isunavailable. When a transplanting into a different a species, it isdesirable to select a host such that the size of the organs in the hostand donor are similar. In addition, the host is selected to minimizetransmission of communicable diseases.

[0026] Autoimmune diseases that may be treated or prevented by thepresent invention include but are not limited to type 1 insulindependant diabetes, inflammatory bowel disease, multiple sclerosis,rheumatoid arthritis, dermatitis, rheumatic fever, Graves Disease,Reynauds Syndrome, chronic active hepatitis, atrophic gastritis,Addisons disease, adult respiratory distress syndrome, meningitis,Sjogren's syndrome, Reiter's syndrome, surcoidosis vasculitis,pernicious anemia, Hashimoto's thyroiditis, celiac disease, ankylosingspondylitis, dermatomyositis, pemphigus, pemphigoid, necrotizingvasculitis, myasthenia gravis, lupus erythematosus, leukocyte adhesiondeficiency, uveitic encephalitis, polymyositis, granulomatosis,glomerulonephritis, progressive systemic sclerosis, psoriatic arthritis,thrombotic thromibocytopenic purpura, primary binary cirrhosisautoimmune haemolytic anemia, autoimmune complications of AIDS, habitualspontaneous abortions, central nervous system inflammatory disorders,antigen -antibody complex mediated diseases, amyotrophic lateralsclerosis, thyroidosis, scleroderma, lupus, and artherosclerosis. Thedisclosed combination therapy is expected to be particularly beneficialwhen used treat type 1 insulin dependant diabetes, inflammatory boweldisease, multiple sclerosis or rheumatoid arthritis.

[0027] A “mammal needing treatment to induce immune tolerance” is amammal with a tissue transplant, a mammal with one of the aforementionedautoimmune diseases or a mammal with an undesired immune response.

[0028] An “effective amount” of an immunosuppressive agent and acompound of Formula (I) is an amount which achieves a degree ofimmunosuppression sufficient to delay, inhibit, suppress or moderatetissue transplant rejection and/or delay, inhibit, suppress or moderateone or more symptoms of an autoimmune disease described herein and/ordelay, inhibit, suppress or moderate an undesired immune response to aforeign antigen such as a therapeutic protein, viral vector, allergen,venom and the like. Preferably, the two agents are used at dosagessuitable for inducing tolerance to the antigen which is the target of anundesired immune response or, alternatively, at dosages at which theimmunosuppressive activities of the two agents synergize. Because manyimmunosuppresive agents described herein and the compound of Formula (I)act synergistically, sub-therapeutic dosages can be used in thedisclosed methods, i.e., dosages which are lower than the amounts thatwould be effective when the agent is used alone. Suitablesub-therapeutic dosages of one or both agents of the disclosedcombination therapy are those which are sufficient, when the two agentsare used in combination, to delay, inhibit, suppress or moderate anundesired immune response to an antigen, as described above. The skilledartisan will be able to determine such dosages using ordinaryexperimentation, such as by using animal models. One advantage of thedisclosed combined therapy, therefore, is that the subject being treatedcan in some instances be spared the side-effects of higher levels ofimmunosuppression resulting from, for example, corticosteroids andinhibitors of calcineurin.

[0029] Suitable dosages of immunosuppressive agents for treating tissuetransplant rejection or autoimmune disease are known to one of ordinaryskill in the art. Suitable dosages of the compound of Formula (I) aretypically those which are described in U.S. patent application Ser. No.09/852,965 and WO 01/87849 to Sneddon et al., “Modulators of TNF-αSignalling”. The entire teachings of these two references areincorporated herein by reference. The dosage to be used is, of course,dependent on various factors such as the mammal to be treated (human orother mammal, weight, sex, age, general health), the severity of thesymptoms, the disorder to be treated, possible accompanying disorders ifany, the nature of the accompanying treatment with otherpharmaceuticals, if any, or the frequency of treatment.

[0030] Typically, the combination therapy according to the inventioncomprises the administration of one to eight individual dosages per dayof about 0.01 mg/Kg/day to about 100 mg/Kg/day of the immunosuppressiveagent and of about 0.01 mg/Kg/day to about 100 mg/Kg/day of a compoundof Formula I. More commonly, about 0.1 mg/Kg/day to about 10 mg/Kg/dayof the immunosuppressive agent and of about 0.1 mg/Kg/day to about 10mg/Kg/day of a compound of Formula (1) are administered and, preferably,about 0.1 mg/Kg/day to about 10 mg/Kg/day of the immunosuppressive agentand of about 0.1 mg/Kg/day to about 1 mg/Kg/day of a compound of Formula(I). The immunosuppressive agents and the compound of Formula (I) can beadministered individually or as a mixture of components in a suitablyformulated pharmaceutical composition.

[0031] The immunosupressive agent and the compound of Formula (I) can beadministered prior to exposure to the antigen causing the immuneresponse, while the subject is being exposed to the antigen, followingexposure to the antigen or any combination thereof.

[0032] The combination preparation according to the invention can alsoinclude combination packs or compositions in which the constituents areplaced side by side and can therefore be administered simultaneously,separately or sequentially to one and the same human or other mammalbody.

[0033] A “mammal” is preferably a human, but can also be an animal inneed of veterinary treatment, e.g., companion animals (e.g., dogs, cats,rabbits and the like), farm animals (e.g., cows, sheep, pigs, horses,and the like) and laboratory animals (e.g., rats, mice, guinea pigs, andthe like).

[0034] Pharmaceutically acceptable salts of the compound of Formula (I)can also used in the disclosed combination therapies. The term“pharmaceutically acceptable salts” refers to salts prepared frompharmaceutically acceptable non-toxic acids. Pharmaceutically acceptablesalts of compound of Formula (I) can be conveniently prepared frompharmaceutically acceptable non-toxic acids, including inorganic andorganic acids, Such acids include for example hydrobromic, hydrochloric,acetic, benzoic, succinic, nitric, sulfuric, benzenesulfuric,camphorsulfonic, ethanesulfonic, citric, fumric, phosphoric,p-toluenesulfonic acid, tantaric, lactic, pamoic, methanesulfonic,maleic, malic, mandelic, gludconic, glutamic, isethionic, mucic,pantothenic and the like.

[0035] Pharmaceutical compositions for use in accordance with thepresent invention thus can be formulated in a conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Techniques forformulation and administration of the components of the instantapplication can be found Remington: The Science and Practice ofPharmacy. 19^(th) edition, March Publishing Co., Easton Pa. (1995).

[0036] Suitable routes of administration can for example include oral,eyedrop, rectal, transmucosal, topical, or intestinal administration;parenternal delivery, including intramuscular, subcutaneous,intramedullary injections as well as intrathecal, directintraventricular intravenous, intraperitoneal, intranasal, orintraocular injections.

[0037] For injection, the agents of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

[0038] For oral administration, the compounds can be formulated readilyby combining the active compounds with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by combining the active compound with a solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients include fillers suchas sugars, (e.g. lactose, sucrose, mannitol, or sorbitol), cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

[0039] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0040] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

[0041] For buccal administration, the compositions may take the form oftablets or lozenges formulated in a conventional manner.

[0042] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of a drypowder inhaler, or an aerosol spray presentation from pressurized packsor a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0043] The compounds can be formulated for parenteral administration byinjection, including bolus injection or continuous infusion.Formulations for injection maybe presented in unit dosage form, such asin ampoules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents.

[0044] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, such as sterile pyrogen-freewater, before use.

[0045] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0046] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Suitableformulations of this type include biocompatible and biodegradablepolymeric hydrogel formulations using crosslinked or water insolublepolysaccharide formulations. Also included are polymerizablepolyethylene oxide formulations. Formulations of this type are disclosedin U.S. Pat. Nos. 5,410,016, 4,713,448, 4,636,524, 6,083,524, 5,785,993,4,937,270 and 5,017,229, the entire teachings of which are incorporatedherein by reference. Such long acting formulations may be administeredby implantation, for example, subcutaneously or intramuscularly or byintramuscular injection. Preferably, they are implanted in themicroenvironment of the transplanted organ or tissue. Thus, for example,the compounds may be formulated with suitable polymeric or hydrophobicmaterials, for example, as an emulsion in an acceptable oil, or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

[0047] The pharmaceutical compositions can also include suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include calcium carbonate, calcium phosphate, various sugars,starches, cellulose derivatives, gelatin, and polymers such aspolyethylene glycols.

[0048] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose. Morespecifically, an effective amount, as previously defined, denotes anamount suitable for inducing tolerance or suppressing an immune responseeffective amounts are described above.

[0049] The term “aryl group”, (e.g., the aryl groups represented by R₁,R₃ and R₄) used alone or as part of a larger moiety such as “aralkyl”(e.g., the aralkyl group represented by R₂), refers to carbocyclicaromatic groups such as phenyl, naphthyl, and anthracyl, and heteroarylgroups such as imidazolyl, isoimidazolyl, thienyl, furanyl, pyridyl,pyrimidyl, pyranyl, pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl,isothiazolyl, oxazolyl, isooxazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl,and tetrazolyl.

[0050] The term “aryl group”, used alone or as part of a larger moietysuch as “aralkyl”, also includes fused polycyclic aromatic ring systemsin which a carbocyclic aromatic ring or heteroaryl ring is fused to oneor more other heteroaryl rings. Examples include benzothienyl,benzofuranyl, indolyl, quinolinyl, benzothiazolyl, quinoxalinyl,quinazolinyl, benzoisothiazolyl, benzooxazolyl, benzoisooxazolyl,benzimidazolyl, quinolinyl, isoquinolinyl and isoindolyl.

[0051] The term “aryl group”, used alone or as part of a larger moietysuch as “aralkyl”, also includes carbocylic aromatic rings or heteroarylaromatic rings fused to a cycloalkyl group or an non-aromaticheterocyclic group. Examples include indanyl, tetrahydronaphthyl andfluorenyl.

[0052] The term “alkyl group” (e.g., the alkyl groups represented byR₁-R₆), used alone or as part of a larger moiety such as “aralkyl”(e.g., the aralkyl group represented by R₂) or “cycloalkylalkyl”, is astraight, branched or cyclic non-aromatic hydrocarbon which iscompletely saturated. Typically, a straight or branched alkyl group hasfrom 1 to about 10 carbon atoms, preferably from 1 to about 4, and acyclic aliphatic group has from 3 to about 10 carbon atoms, preferablyfrom 3 to about 8. Examples of suitable straight or branched alkyl groupinclude methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,tert-butyl, pentyl, hexyl, heptyl or octyl; and examples of suitablecycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl. A C1-C10 straight or branchedalkyl group or a C3-C8 cyclic alkyl group are also referred to as a“lower alkyl” group.

[0053] An “aralkyl group” is an alkyl group substituted with one or morearyl groups. A “substituted aralkyl group” can have one or moresubstituents on the alkyl part of the aralkyl group and/or on the arylpart of the aralkyl group. Suitable aralkyl group substituents aredescribed below in the section providing examples of aryl groupsubstituents and alkyl group substituents.

[0054] A “heteroaralkyl group” is an alkyl group substituted with one ormore heteroaryl groups. A “substituted heteroaralkyl group” can have oneor more substituents on the alkyl part of the heteroaralkyl group and/oron the heteroaryl part of the heteroaralkyl group. Suitableheteroaralkyl group substituents are described below in the sectionproviding examples of aryl group substituents and alkyl groupsubstituents.

[0055] A “cycloalkylalkyl group” is an alkyl group substituted with oneor more cycloalkyl groups. A “substituted cycloalkylalkyl group” canhave one or more substituents on the alkyl part or cycloalkylalkyl partof the cycloalkylalkyl group. Suitable cycloalkylalkyl groupsubstituents are described below in the section providing examples ofalkyl group substituents.

[0056] A “heterocycloalkylalkyl group” is an alkyl group substitutedwith one or more non-aromatic heterocyclic groups. A “substitutedheterocycloalkylalkyl group” can have one or more substituents on thealkyl part or non-aromatic heterocyclic part of theheterocycloalkylalkyl group. Suitable heterocycloalkylalkyl groupsubstituents are described below in the section providing examples ofalkyl group substituents and non-aromatic heterocyclic groupsubstitutents.

[0057] The term “non-aromatic heterocyclic ring” (e.g., non-aromaticheterocyclic groups represented by —NR₅R₆) refers to non-aromatic ringsystems typically having five to fourteen members, preferably five toten, in which one or more ring carbons, preferably one to four, are eachreplaced by a heteroatom such as N, O, or S. Examples of non-aromaticheterocyclic rings include 3-1H-benzimidazol-2-one, 3-tetrahydrofuranyl,2-tetrahydropyranyl, 3-tetrahydropyranyl, 4-tetrahydropyranyl,[1,3]-dioxalanyl, [1,3]-dithiolanyl, [1,3]-dioxanyl,2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholinyl,3-morpholinyl, 4-morpholinyl, 2-thiomorpholinyl, 3-thiomorpholinyl,4-thiomorpholinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrorolidinyl,1-piperazinyl, 2-piperazinyl, 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl, diazolonyl, N-substituteddiazolonyl, 1-pthalimidinyl, benzoxanyl, benzopyrrolidinyl,benzopiperidinyl, benzoxolanyl, benzothiolanyl, and benzothianyl.

[0058] Suitable substituents for an alkyl group, for a carbon atom on anaryl group or a non-aromatic heterocyclic group are those which do notsubstantially interfere with the ability of the compound to inhibittransplant rejection. Examples of suitable substituents for a carbonatom of an aryl, alkyl or a carbon atom of a non-aromatic heterocyclicgroup include —OH, halogen (—Br, —Cl, —I and —F), R, —CH₂R, —OCH₂R,—CH₂OC(O)R, —OR, —O—COR, —COR, —CN, —NO₂, —COOH, —SO₃H, —NH₂, —NHR,—N(R)₂, —COOR, —CHO, —CONH₂, —CONHR, —CON(R)₂, —NHCOR, —NRCOR, —NHCONH₂,—NHCONRH, —NHCON(R)₂, —NRCONH₂, —NRCONRH, —NRCON(R)₂, —C(═NH)—NH₂,—C(═NH)—NHR, —C(═NH)—N(R)₂, —C(═NR)—NH₂, —C(═NR)—NHR, —C(═NR)—N(R)₂,—NH—C(═NH)—NH₂, —NH—C(═NH)—NHR, —NH—C(═NH)—N(R)₂, —NH—C(═NR)—NH₂,—NH—C(═NR)—NHR, —NH—C(═NR)—N(R)₂, —NRH—C(═NH)—NH₂, —NR—C(═NH)—NHR,—NR—C(═NH)—N(R)₂, —NR—C(═NR)—NH₂, —NR—C(═NR)—NHR, —NR—C(═NR)—N(R)₂,—SO₂NH₂, —SO₂NHR, —SO₂NR₂, —SH, —SO_(k)R (k is 0, 1 or 2) and—NH—C(═NH)—NH₂. Each R is independently an alkyl, substituted alkyl,benzyl, substituted benzyl, aryl or substituted aryl group. Preferably,R is an alkyl, benzylic or aryl group. In addition, —N(R)₂, takentogether, can also form a substituted or unsubstituted non-aromaticheterocyclic group, such as pyrollidinyl, piperidinyl, morpholinyl andthiomorpholinyl. Examples of substituents on the alkyl, aryl or benzylgroup represented by R include amino, alkylamino, dialkylamino,aminocarbonyl, halogen, alkyl, alkylaminocarbonyl,dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl,alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl. A substituted aryl,alkyl and non-aromatic heterocyclic group can have more than onesubstitutent.

[0059] Suitable substitutents on the nitrogen of a non-aromaticheterocyclic group or heteroaryl group include —R′, —N(R′)₂, —C(O)R′,—CO₂ R′, —C(O)C(O)R′, —C(O)CH₂ C(O)R′, —SO₂R′, —SO₂ N(R′)₂,—C(═S)N(R′)₂, —C(═NH)—N(R′)₂, and —NR′SO₂R′; wherein R′ is hydrogen, analkyl group, a substituted alkyl group, phenyl (Ph), substituted Ph,—O(Ph), substituted —O(Ph), CH₂(Ph), or an unsubstituted heteroaryl ornon-aromatic heterocyclic ring. Examples of substituents on thealiphatic group or the phenyl ring represented by R′ include amino,alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl,alkylaminocarbonyl, dialkylaminocarbonyloxy, alkoxy, nitro, cyano,carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, orhaloalkyl.

[0060] The synthesis of the compound of Formula (I) can be accomplishedusing methodologies disclosed in WO 01/87849 and U.S. patent applicationSer. No. 09/852,965 previously incorporated by reference in theirentirety.

[0061] In a preferred embodiment, the variables for the compoundrepresented by Formula (I) are as provided above, provided that thecompound is characterized by one or more of the following features:

[0062] a) R₁ is an optionally substituted aryl group or an optionallysubstituted C₁-C₄ aralkyl group.

[0063] b) R₁ is an optionally substituted phenyl group or an optionallysubstituted phenyl-C₁-C₄ alkyl group. Preferred substituents for thesevalues of R₁ include C₁-C₄ alkyl, C₁-C₄ alkoxy, halogen, CN,C₁-C₄-alkylthiol, C₁-C₄-haloalkyl and phenoxy.

[0064] c) R₂ is an optionally substituted heteroaralkyl group or analkyl group substituted with —NR₅R₆.

[0065] d) R₂ is an optionally substituted imadazolyl-C₁-C₄-alkyl groupor a C₁-C₄ alkyl group substituted with —NR₅R₆.

[0066] e) R₂ is an optionally substituted 2-(imidazol-4-yl)ethyl, anoptionally substituted 3-(imidazol-4-yl)propyl, an optionallysubstituted 3-(imidazol-1-yl)propyl, an optionally substituted2-(morpholin-4-yl)ethyl, an optionally substituted 2-(4-pyrazolyl)ethyl,an optionally substituted 2-N,N-dimethylaminoethyl or an optionallysubstituted 3-N,N-dimethylaminopropyl. Preferably,R₂2-(imidazol-4-yl)ethyl.

[0067] f) R₃ is an optionally substituted aryl group or an optionallysubstituted C₁-C₄ aralkyl group.

[0068] g) R₃ is an optionally substituted an optionally substituted2-cyclohexylethyl, an optionally substituted 2-cyclopentylethyl, or anoptionally substituted C₃-C₈ secondary or tertiary alkyl group.

[0069] h) R₃ is an optionally substituted phenyl, an optionallysubstituted phenyl-C₁-C₄-alkyl, an optionally substituteddiphenyl-C₁-C₄-alkyl, an optionally substituted pyrazolyl, an optionallysubstituted pyrazolyl-C₁-C₄-alkyl, an optionally substituted indolyl, anoptionally substituted indolyl-C₁-C₄-alkyl, thienylphenyl,thienylphenyl-C₁-C₄-alkyl, furanylphenyl, furanylphenyl-C₁-C₄-alkyl, anoptionally substituted fluorenyl, an optionally substitutedfluorenyl-C₁-C₄-alkyl, an optionally substituted naphthyl, an optionallysubstituted naphthyl-C₁-C₄-alkyl, an optionally substitutedquinoxalinyl, an optionally substituted quinoxalinyl-C₁-C₄-alkyl, anoptionally substituted quinazolinyl, an optionally substitutedquinazolinyl-C₁-C₄-alkyl, an optionally substituted pyrolyl, anoptionally substituted pyrolyl-C₁-C₄-alkyl, an optionally substitutedthienyl, an optionally substituted thienyl-C₁-C₄-alkyl, an optionallysubstituted furanyl, an optionally substituted furanyl-C₁-C₄-alkyl, anoptionally substituted pyridyl or an optionally substituted-C₁-C₄pyridyl.

[0070] i) R₃ is represented by Formula (II):

[0071] Ring A is substituted or unsubstituted; R₇ is an optionallysubstituted phenyl, optionally substituted furanyl, optionallysubstituted thienyl or optionally substituted pyridyl group; n is aninteger from 1-4; and X is a bond, CH₂, OCH₂, CH₂OC(O), CO, OC(O),C(O)O, O, S, SO or SO₂. Examples of suitable substituents for Ring A andthe aryl groups represented by R₇ are provided in section abovedescribing suitable aryl group substituents.

[0072] j) R₃ is represented by Formula (II), wherein Ring A issubstituted or unsubstituted; R₇ is an optionally substituted phenylgroup; n is 1; and X is CO.

[0073] k) R₃ is represented by Formula (II), Ring A is unsubstituted R₇is an optionally substituted phenyl group; n is 1; and X is CO. Morepreferably, R₇ is a phenyl group.

[0074] l) R₄ is an optionally substituted aryl group, an optionallysubstituted cycloalkyl group, an optionally substituted C₁-C₄ aralkylgroup or an optionally substituted C₁-C₄ cycloalkylalkyl group.

[0075] m) R₄ is an optionally substituted phenyl group, an optionallysubstituted phenyl-C₁-C₄-alkyl group, an optionally substituteddiphenyl-C₁-C₄-alkyl group, an optionally substitutedC₃-C₈-cycloalkyl-C₁-C₄-alkyl group or an optionally substituteddi-(C₃-C₈-cycloalkyl)-C₁-C₄-alkyl group.

[0076] n) R₄ is an optionally substituted benzyl, an optionallysubstituted diphenylmethyl, an optionally substituted 2-phenylethyl, anoptionally substituted 1,2-diphenylethyl, an optionally substituted2,2-diphenylethyl or an optionally substituted 3,3-diphenylpropyl.Preferred substituents for these values of R₄ include C₁-C₄ alkyl, C₁-C₄alkoxy, halogen, CN, C₁-C₄-alkylthiol, C₁-C₄-haloalkyl and phenoxy.

[0077] In another preferred embodiment, the compound of Formula (I) ischaracterized by one or more of Features a), f) and 1), and preferablyall of Features a), f) and 1).

[0078] In another preferred embodiment, the compound of Formula (I) ischaracterized by one or more of Features b), h) and m), and preferablyall of Features b), h) and m). More preferably, the compound of Formula(I) is additionally characterized by Feature d).

[0079] In another preferred embodiment, the compound of Formula (I) ischaracterized by one or more of Features b), d), i) and m), andpreferably all of Features b), d), i) and m). Alternatively, Feature m)is replaced by Feature n). In another alternative, Feature m) isreplaced by Feature n) and Feature i) is replaced by Feature j).

[0080] In another preferred embodiment, the compound of Formula (I) ischaracterized by one or more of Features b), d), k) and m), andpreferably all of Features b), d), k) and m).

[0081] In yet another embodiment, the method of the present invention iscarried out with the compound of Formula (III) in place of the compoundof Formula (I):

[0082] R₁₁ is —H, a substituted or unsubstituted aryl, a substituted orunsubstituted aralkyl, a substituted or unsubstituted heteroaryl or asubstituted or unsubstituted heteroaralkyl;

[0083] R₁₂ is alkyl substituted with NR₁₅R₁₆, a substituted orunsubstituted aryl, a substituted or unsubstituted heteroaralkyl, or asubstituted or unsubstituted heterocycloalkylalkyl;

[0084] R₁₃ is a substituted or unsubstituted alkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted aralkyl, asubstituted or unsubstituted cycloalkylalkyl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted heteroaralkyl,a substituted or unsubstituted benzophenonyl, or a substituted orunsubstituted cycloalkylalkyl; and

[0085] each R₁₄ is independently, —H, a substituted or unsubstitutedalkyl, a substituted or unsubstituted aryl, substituted or unsubstitutedaralkyl or a substituted or unsubstituted heteroaralkyl;

[0086] R₁₅ and R₁₆ are independently selected from H, a substituted orunsubstituted alkyl, a substituted or unsubstituted cycloalkyl, asubstituted or unsubstituted aryl or unsubstituted aralkyl or R₁₅ andR₁₆ together with the nitrogen to which they are attached are aheterocycloalkyl.

[0087] The invention is illustrated by the following examples, which arenot intended to be limiting in any way.

EXEMPLIFICATION Example 1 Synergistic Suppression of TransplantRejection Using a Combination Therapy of Compound 1 and Rapamycin in aMouse Model

[0088] The ability of a combination therapy of rapamycin and Compound 1to inhibit transplant rejection in a mouse model was tested.Specifically, the heart from C57/BL6 mice was transplanted into Balb/crecipient mice (total MHC mismatch) using standard protocol described inHancock, W. W., et al. and Yuan et al., Transplantation 73: 1736,Proc.Natl. Acad. Sci. USA 93: 13967 (1996), the entire teachings ofwhich are incorporated herein by reference. Following surgery, the micewere divided into the following treatment groups:

[0089] Group I is a control group that was untreated.

[0090] Group II was treated with Compound 1 at a dose of 75 mg/kg/daysubcutaneously for fourteen days following surgery.

[0091] Group III was treated with rapamycin at a dose of 0.3 mg/kg/dayintraperitoneally for three days following surgery.

[0092] Group IV was treated with Compound 1 at a dose of 75 mg/kg/dayintraperitoneally for fourteen days following surgery and with rapamycinat a dose of 0.3 mg/kg/day intraperitoneally for three days followingsurgery. It is noted that this dose of rapamycin is about ten timeslower than is typically used to suppress transplant rejection.

[0093] The results are shown in FIG. 1. As can be seen, control micereceiving no treatment reproducibly experienced rejection of theirtransplanted hearts after only ten days. Mice receiving rapamycin aloneor the Compound 1 alone showed a modest (two to three weeks) butstatistically significant delay (p<0.001) in the onset of rejection.However, mice receiving the combination therapy of Compound 1 andrapamycin had 100% graft survival for over 100 days, indicating that thetwo drugs acted synergistically (p<0.001). In fact, the animals treatedin this manner never showed signs of acute rejection and were sacrificedat 120 days following surgery so that the transplanted heart couldanalyzed histologically.

Example 2 Synergistic Suppression of Transplant Rejection Using aCombination Therapy of Compound 1 and Anti-CD40L in a Mouse Model

[0094] The ability of a combination therapy of anti CD40L and Compound 1to inhibit transplant rejection in a mouse model was tested according tothe protocol of Example 1 using a single 250 μg dose of anti CD40Ladministered intraperitoneally in place of rapamycin. The results areshown in FIG. 2. As can be seen, mice receiving the combination therapyhad 100% graft survival for over 100 days, indicating that the two drugsacted synergistically (p<0.001). Again, the animals treated in thismanner never showed signs of acute rejection and were sacrificed at 120days following surgery so that the transplanted heart could analyzedhistologically.

Example 3 Induction of Specific Immune Tolerance Using a CombinationTherapy of the Compound 1 and Rapamycin in a Mouse Model

[0095] The ability of a combination therapy of rapamycin and theCompound 1 to induce specific immune tolerance in a mouse model wastested. Specifically, the heart from C57/BL6 mice was transplanted intoBalb/c recipient mice (total MHC mismatch) using standard protocoldescribed in Hancock, W. W., et al. Proc. Natl. Acad. Sci. USA 93: 13967(1996) and and Yuan et al., Transplantation 73:1736. Following surgery,the mice were treated with the Compound 1 at a dose of 75 mg/kg/dayintraperitoneally for fourteen days and with rapamycin at a dose of 0.3mg/kg/day intraperitoneally for three days. Following treatment, themice were divided into two groups of 2 mice each. The first groupreceived a second transplanted heart from the same donor strain of mice;mice from the second group received a second heart transplant from anunrelated third party donor. The hearts from the mice in the first groupwere not rejected, whereas the all of the mice in second group rejectedthe new hearts. This indicates that the combination therapy blinds theimmune system to the alloantigen immediately at hand for an extendedperiod but does not result in non-specific immune ablation.

Example 4 Effect of a Combination Therapy of the Compound 1 andCyclosporin A in a Mouse Model

[0096] The ability of a combination therapy of cyclosporin A andCompound 1 to inhibit transplant rejection in a mouse model was testedaccording to the protocol of Example 1 using 10 mg/kg/day of cyclosporinA administered intraperitoneally in place of rapamycin. It was observedthat the combination of Compound 1 and cyclosporin A appeared not to besynergistic or additive with respect to prolongation of allograftsurvival.

What is claimed is:
 1. A method of inhibiting rejection of atransplanted organ, tissue or cell in a mammal, the method comprisingthe step of administering to the recipient mammal an effective amount ofan immunosuppressive agent and an effective amount of a compoundrepresented by the following structural formula:

or a physiological salt thereof, wherein: R₁ is a substituted orunsubstituted aryl group or a substituted or unsubstituted alkyl group;R₂ is an optionally substituted aralkyl group or an alkyl groupsubstituted with —NR₅R₆; R₃ is a substituted or unsubstituted alkylgroup or a substituted or unsubstituted aryl group; R₄ a substituted orunsubstituted alkyl group or a substituted or unsubstituted aryl group;and R₅ and R₆ are independently selected from a substituted orunsubstituted alkyl group or a substituted or unsubstituted aryl groupor R₅ and R₆ taken together with the nitrogen to which they are attachedare a non-aromatic heterocyclic group.
 2. The method of claim 1 whereinthe transplanted organ, tissue or cell is a transplanted heart, kidney,lung, liver, pancreas, skin or bone marrow.
 3. The method of claim 1wherein the mammal is the recipient of a transplanted stem cell(s). 4.The method of claim 1 wherein the transplanted organ, tissue or cell isxenogenic or bio-engineered.
 5. The method of claim 1 wherein theimmunosuppressive agent is an anti lymphocyte antibody.
 6. The method ofclaim 1 wherein the immunosuppressive agent is an anti-CD40L monoclonalantibody or rapamycin.
 7. The method of claim 1 wherein R₂ is anoptionally substituted heteroaralkyl group or an alkyl group substitutedwith —NR₅R₆.
 8. The method of claim 7 wherein: a) R₁ is an optionallysubstituted aryl group or an optionally substituted C₁-C₄ aralkyl group;b) R₃ is an optionally substituted aryl group or an optionallysubstituted C₁-C₄ aralkyl group; and c) R₄ is an optionally substitutedaryl group, an optionally substituted cycloalkyl group, an optionallysubstituted C₁-C₄ aralkyl group or an optionally substituted C₁-C₄cycloalkylalkyl group.
 9. The method of claim 7 wherein: a) R₁ is anoptionally substituted phenyl group or an optionally substitutedphenyl-C₁-C₄ alkyl group; b) R₃ a substituted or unsubstituted phenyl,phenyl-C₁-C₄-alkyl, diphenyl-C₁-C₄-alkyl, pyrazolyl,pyrazolyl-C₁-C₄-alkyl, indolyl, indolyl-C₁-C₄-alkyl, thienylphenyl,thienylphenyl-C₁-C₄-alkyl, furanylphenyl, furanylphenyl-C₁-C₄-alkyl,fluorenyl, fluorenyl-C₁-C₄-alkyl, naphthyl, naphthyl-C₁-C₄-alkyl,quinoxalinyl, quinoxalinyl-C₁-C₄-alkyl, an optionally substitutedquinazolinyl, an optionally substituted quinazolinyl-C₁-C₄-alkyl,pyrolyl, pyrolyl-C₁-C₄-alkyl, thienyl, thienyl-C₁-C₄-alkyl, furanyl orfuranyl-C₁-C₄-alkyl; and c) R₄ is an optionally substituted phenylgroup, an optionally substituted phenyl-C₁-C₄-alkyl group, an optionallysubstituted diphenyl-C₁-C₄-alkyl group, an optionally substitutedC₃-C₈-cycloalkyl-C₁-C₄-alkyl group or an optionally substituteddi-(C₃-C₈-cycloalkyl)-C₁-C₄-alkyl group.
 10. The method of claim 9wherein R₂ is an optionally substituted imadazolyl-C₁-C₄-alkyl group ora C₁-C₄ alkyl group substituted with —NR₅R₆.
 11. The method of claim 10wherein: R₁ is a phenyl group or phenyl-C₁-C₄ alkyl group eachoptionally substituted with R, —CH₂R, —OCH₂R, —CH₂OC(O)R, —OH, halogen,—OR, —O—COR, —COR, —CN, —NO₂, —COOH, —SO₃H, —NH₂, —NHR, —N(R)₂, —COOR,—CHO, —CONH₂, —CONHR, —CON(R)₂, —NHCOR, —NRCOR, —NHCONH₂, —NHCONRH,—NHCON(R)₂, —NRCONH₂, —NRCONRH, —NRCON(R)₂, —C(═NH)—NH₂, —C(═NH)—NHR,—C(═NH)—N(R)₂, —C(═NR)—NH₂, —C(═NR)—NHR, —C(═NR)—N(R)₂, —NH—C(═NH)—NH₂,—NH—C(═NH)—NHR, —NH—C(═NH)—N(R)₂, —NH—C(═NR)—NH₂, —NH—C(═NR)—NHR,—NH—C(═NR)—N(R)₂, —NRH—C(═NH)—NH₂, —NR—C(═NH)—NHR, —NR—C(═NH)—N(R)₂,—NR—C(═NR)—NH₂, —NR—C(═NR)—NHR, —NR—C(═NR)—N(R)₂, —SO₂NH₂, —SO₂NHR,—SO₂N(R)₂, —SH or-SokR; R₃ is represented by the following structuralformula:

R₄ is 2,2-diphenylethyl, 2-phenylethyl, benzyl, diphenylmethyl,1,2-diphenylethyl, 3,3-diphenylpropyl, benzyl, or 2-pyridylethyl, eachoptionally substituted with —OH, halogen, R, —CH₂R, —OCH₂R, —CH₂OC(O)R,—OR, —O—COR, —COR, —CN, —NO₂, —COOH, —SO₃H, —NH₂, —N(R)₂, —COOR, —CHO,—CONH₂, —CONHR, —CON(R)₂, —NHCOR, —NRCOR, —NHCONH₂, —NHCONRH,—NHCON(R)₂, —NRCONH₂, —NRCONRH, —NRCON(R)₂, —C(═NH)—NH₂, —C(═NH)—NHR,—C(═NH)—N(R)₂, —C(═NR)—NH₂, —C(═NR)—NHR, —C(═NR)—N(R)₂, —NH—C(═NH)—NH₂,—NH—C(═NH)—NHR, —NH—C(═NH)—N(R)₂, —NH—C(═NR)—NH₂, —NH—C(═NR)—NHR,—NH—C(═NR)—N(R)₂, —NRH—C(═NH)—NH₂, —NR—C(═NH)—NHR, —NR—C(═NH)—N(R)₂,—NR—C(═NR)—NH₂, —NR—C(═NR)—NHR, —NR—C(═NR)—N(R)₂, —SO₂NH₂, —SO₂NHR,—SO₂N(R)₂, —SH or —SOkR; Ring A substituted or unsubstituted; R₇ is anoptionally substituted phenyl, furanyl, thienyl or pyridyl group; n isan integer from 1-4; and X is a bond, CH₂, OCH₂, CH₂OC(O), CO, OC(O),C(O)O, O, S, SO or SO₂; each R is independently C₁-C₄ alkyl or phenyloptionally substituted with amino, alkylamino, dialkylamino,aminocarbonyl, halogen, alkyl, alkylaminocarbonyl,dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl,alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl; and k is zero, one ortwo.
 12. The method of claim 11 wherein R₁ is a phenyl group orphenyl-C₁-C₂ alkyl group, each optionally substituted with C₁-C₄ alkyl,C₁-C₄ alkoxy, halogen, CN, C₁-C₄-alkylthiol, C₁-C₄-haloalkyl or phenoxy;R₄ is 2,2-diphenylethyl, 2-phenylethyl, benzyl, diphenylmethyl,1,2-diphenylethyl, 3,3-diphenylpropyl, benzyl, or 2-pyridylethyl, eachoptionally substituted with C₁-C₄ alkyl, C₁-C₄ alkoxy, halogen, CN,C₁-C₄-alkylthiol, C₁-C₄-haloalkyl or phenoxy; R₇ is an optionallysubstituted phenyl group; n is 1; and X is CO.
 13. The method of claim12 wherein Ring A is unsubstituted and R₇ is a phenyl group optionallysubstituted with R, —CH₂R, —OCH₂R, —CH₂OC(O)R, —OH, halogen, —OR,—O—COR, —COR, —CN, —NO₂, —COOH, —SO₃H, —NH₂, —NHR, —N(R)₂, —COOR, —CHO,—CONH₂, —CONHR, —CON(R)₂, —NHCOR, —NRCOR, —NHCONH₂, —NHCONRH,—NHCON(R)₂, —NRCONH₂, —NRCONRH, —NRCON(R)₂, —C(═NH)—NH₂, —C(═NH)—NHR,—C(═NH)—N(R)₂, —C(═NR)—NH₂, —C(═NR)—NHR, —C(═NR)—N(R)₂, —NH—C(═NH)—NH₂,—NH—C(═NH)—NHR, —NH—C(═NH)—N(R)₂, —NH—C(═NR)—NH₂, —NH—C(═NR)—NHR,—NH—C(═NR)—N(R)₂, —NRH—C(═NH)—NH₂, —NR—C(═NH)—NHR, —NR—C(═NH)—N(R)₂,—NR—C(═NR)—NH₂, —NR—C(═NR)—NHR, —NR—C(═NR)—N(R)₂, —SO₂NH₂, —SO₂NHR,—SO₂N(R)₂, —SH or —SO_(k)R.
 14. The method of claim 13 wherein R₇ is aphenyl group; and R₂ is 2-(imidazol-4-yl)ethyl.
 15. A method ofinhibiting rejection of a transplanted organ, tissue or cell in amammal, the method comprising the step of administering to the recipientmammal an effective amount of an anti CD40L monoclonal antibody orrapamycin and an effective amount of a compound represented by thefollowing structural formula:

or a pharmaceutically acceptable salt of the compound.
 16. The method ofclaim 15 wherein the transplanted organ, tissue or cell is atransplanted heart, kidney, lung, liver, pancreas, skin or bone marrow.17. A composition comprising an immunsuppressive agent and a compoundrepresented by the following structural formula:

or a physiological salt thereof, wherein: R₁ is a substituted orunsubstituted aryl group or a substituted or unsubstituted alkyl group;R₂ is an optionally substituted aralkyl group or an alkyl groupsubstituted with —NR₅R₆; R₃ is a substituted or unsubstituted alkylgroup or a substituted or unsubstituted aryl group; R₄ a substituted orunsubstituted alkyl group or a substituted or unsubstituted aryl group;and R₅ and R₆ are independently selected from a substituted orunsubstituted alkyl group or a substituted or unsubstituted aryl groupor R₅ and R₆ taken together with the nitrogen to which they are attachedare a non-aromatic heterocyclic group.
 18. The composition of claim 17wherein the immunosuppressive agent is an anti CD40L monoclonal antibodyor repamycin.
 19. A composition comprising an anti CD40L monoclonalantibody or repamycin and a compound represented by the followingstructural formula:

or a pharmaceutically acceptable salt of the compound.

R₁₁ is —H, a substituted or unsubstituted aryl, a substituted orunsubstituted aralkyl, a substituted or unsubstituted heteroaryl or asubstituted or unsubstituted heteroaralkyl; R₁₂ is alkyl substitutedwith NR₁₅R₁₆, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaralkyl, or a substituted or unsubstitutedheterocycloalkylalkyl; R₁₃ is a substituted or unsubstituted alkyl, asubstituted or unsubstituted aryl, a substituted or unsubstitutedaralkyl, a substituted or unsubstituted cycloalkylalkyl, a substitutedor unsubstituted heteroaryl, a substituted or unsubstitutedheteroaralkyl, a substituted or unsubstituted benzophenonyl, or asubstituted or unsubstituted cycloalkylalkyl; and each R₁₄ isindependently, —H, a substituted or unsubstituted alkyl, a substitutedor unsubstituted aryl, substituted or unsubstituted aralkyl or asubstituted or unsubstituted heteroaralkyl; R₁₅ and R₁₆ areindependently selected from H, a substituted or unsubstituted alkyl, asubstituted or unsubstituted cycloalkyl, a substituted or unsubstitutedaryl or unsubstituted aralkyl or R₁₅ and R₁₆ together with the nitrogento which they are attached are a heterocycloalkyl.
 20. A method ofinhibiting rejection of a transplanted organ, tissue or cell in amammal, the method comprising the step of administering to the recipientmammal an effective amount of an immunosuppressive agent and aneffective amount of a compound represented by the following structuralformula:

R₁₁ is —H, a substituted or unsubstituted aryl, a substituted orunsubstituted aralkyl, a substituted or unsubstituted heteroaryl or asubstituted or unsubstituted heteroaralkyl; R₁₂ is alkyl substitutedwith NR₁₅R₁₆, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaralkyl, or a substituted or unsubstitutedheterocycloalkylalkyl; R₁₃ is a substituted or unsubstituted alkyl, asubstituted or unsubstituted aryl, a substituted or unsubstitutedaralkyl, a substituted or unsubstituted cycloalkylalkyl, a substitutedor unsubstituted heteroaryl, a substituted or unsubstitutedheteroaralkyl, a substituted or unsubstituted benzophenonyl, or asubstituted or unsubstituted cycloalkylalkyl; and each R₁₄ isindependently, —H, a substituted or unsubstituted alkyl, a substitutedor unsubstituted aryl, substituted or unsubstituted aralkyl or asubstituted or unsubstituted heteroaralkyl; R₁₅ and R₁₆ areindependently selected from H, a substituted or unsubstituted alkyl, asubstituted or unsubstituted cycloalkyl, a substituted or unsubstitutedaryl or unsubstituted aralkyl or R₁₅ and R₁₆ together with the nitrogento which they are attached are a heterocycloalkyl.